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. 2009 Sep 24;35(6):889-902.
doi: 10.1016/j.molcel.2009.09.011.

Interaction of transcriptional regulators with specific nucleosomes across the Saccharomyces genome

R Thomas Koerber  1 , Ho Sung Rhee, Cizhong Jiang, B Franklin Pugh
Affiliations

Interaction of transcriptional regulators with specific nucleosomes across the Saccharomyces genome

R Thomas Koerber et al. Mol Cell. .

Abstract

A canonical nucleosome architecture around promoters establishes the context in which proteins regulate gene expression. Whether gene regulatory proteins that interact with nucleosomes are selective for individual nucleosome positions across the genome is not known. Here, we examine on a genomic scale several protein-nucleosome interactions, including those that (1) bind histones (Bdf1/SWR1 and Srm1), (2) bind specific DNA sequences (Rap1 and Reb1), and (3) potentially collide with nucleosomes during transcription (RNA polymerase II). We find that the Bdf1/SWR1 complex forms a dinucleosome complex that is selective for the +1 and +2 nucleosomes of active genes. Rap1 selectively binds to its cognate site on the rotationally exposed first and second helical turn of nucleosomal DNA. We find that a transcribing RNA polymerase creates a delocalized state of resident nucleosomes. These findings suggest that nucleosomes around promoter regions have position-specific functions and that some gene regulators have position-specific nucleosomal interactions.

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Figures

Figure 1
Figure 1. Identification of Factor-nucleosome Interactions in vivo
(A) Diagram outlining the factor-nucleosome interaction assay. Cells encoding a TAP-tagged protein are treated with formaldehyde. Chromatin is then isolated and solubilized to mononucleosomes with MNase, then subjected to TAP purification. Adapter capture of mono-nucleosomal DNA produces an ~200 bp LM-PCR product, which can be subsequently mapped genome-wide. (B) LM-PCR detection of the indicated factor-nucleosome interaction. BY4741 is a negative control that lacks the TAP tag; H2A.Z is a positive control. Input represents the equivalent of 10−5 ml of cell culture used in LM-PCR, and is the material used for the immunoprecipitation. The volume of cell culture (at OD600 = 0.8) used in the TAP purification is indicated. (C) Bdf1-nucleosome interactions are dependent on Nua4 (Esa1)-directed acetylation. LM-PCR experiments were performed on the indicated strains as described in panels A and B.
Figure 2
Figure 2. SWR1/Bdf1 are Enriched at the +1 and +2 Nucleosomes
(A) Distribution of sequence tags for Bdf1-bound nucleosomes and all nucleosomes in a representative section of the genome. The upper panel (cluster 1) displays the enrichment at the +1 nucleosome, and the bottom panel (cluster 2) displays the enrichment at the +2 nucleosome. (B) Same as panel A, for a collection of loci in which the tag counts have been binned (25 bp bins, and smoothed via a 3-bin moving average) and converted to color intensities. Each gene is represented by three tracks (Bdf1-bound nucleosomes in blue, Vps72-bound nucleosomes in gold, and all nucleosomes in gray). Genes are aligned by their transcriptional start site (TSS) (David et al., 2006). TSS (green lines) and transcript termination sites (TTS, red lines) in the region are shown. Transcription frequency (mRNA/hr) (Holstege et al., 1998) is shown as horizontal red bars, with 10 mRNA/hr indicated by the dashed line. (C) A composite distribution of tags around the TSS for those genes having the top 150 Bdf1-bound nucleosomes (blue trace). Also shown for the same set of genes are tag distributions for nucleosomes bound by Vps72 (gold trace), H2A.Z (cyan fill), and H3/H4 (“All”, in gray fill). (D) Venn diagram of the overlap of genes having significant (P < 0.05) Bdf1- or Vps72-interacting nucleosomes.
Figure 3
Figure 3. Bdf1 Forms a Di-nucleosome Complex with the +1 and +2 Nucleosomes
(A) LM-PCR of Bdf1-bound di-nucleosomes. The assay was performed as in Fig. 1A, except that formaldehyde crosslinking was omitted and less chaotropic extraction buffers were used (termed “CoIP”). (B) Bdf1 di-nucleosome material from panel A was analyzed by Affymetrix high density tiling arrays. The distribution of the highest 4,722 (P <0.05) peaks, representing the di-nucleosome midpoint, around the nearest TSS is shown. (C) Model of how binding of Bdf1 to acetylated +1/+2 nucleosomes might promote H2A.Z incorporation via the SWR1 complex. The asterisks represent histone acetylation marks. “Z” denotes H2A.Z. (D) Illustration of how the model in Fig. 3C might allow Pol II to traverse the region and maintain histone modification states. (E,F) Distribution of Pol II (produced by standard sonication-based ChIP) (Venters and Pugh, 2009) and Pol II-bound nucleosomes around the TSS. The Bdf1-bound filled trace is from Fig. 2C. Panels B and C are for the same genes as in Fig. 2C. (G) Nucleosome exchange rate of Bdf1-bound nucleosomes. Nucleosome exchange rates were from (Dion et al., 2007; Rufiange et al., 2007). Exchange rates for the top and bottom 5 percentile at the +1/+2 nucleosomal positions, along with the median value for the top 150 Bdf1-bound genes +1/+2 nucleosomes, were divided by the genome-wide median for +1/+2, then log10 transformed and plotted.
Figure 4
Figure 4. Rap1 Associates with a Specific Rotational Setting on the −1 Nucleosome
(A–C), Distribution of sequence tags for Rap1-bound nucleosomes. The panels show A) individual loci, B) a collection of genes, C) and a composite profile of tags, as described in Fig. 2. Also illustrated in C is a Rap1-unallowable (X) and –allowable (√) promoter nucleosome configuration. (D) Distribution of Rap1 binding motifs on the −1 nucleosome. Plotted is the midpoint of the 13-bp motif (ACACCCRYACAYM for the top 150 Rap1-bound nucleosomes), which is composed of two separate half-sites as illustrated by the blue barbells. The rotational setting of DNA on the histone surface is indicated above the plot, where black indicates that the major groove faces inward. Motifs that existed as tandem repeats (found near telomeres) were removed and plotted separately (green trace). Also shown is the distribution of Rap1 sites that have previously been shown to be bound by Rap1 (Buck and Lieb, 2006), but were detected here as having insignificant (P >0.3) Rap1-nucleosomal interactions (red trace). (E) Model depicting the interaction of Rap1 with the nucleosome core particle in the rotational and translational setting defined in vivo. The model represents a merge of the Rap1/DNA structure (blue/red, 1IGN) with a portion of the nucleosome core particle structure (gray, 1KX5).
Figure 5
Figure 5. Reb1 Associates Specifically with the −1 Nucleosome
Panel descriptions are as indicated in Fig. 4, but for Reb1. Panel D shows the frequency distribution of nucleosomal DNA lengths as deviations from the canonical 147 bp, for Reb1-bound, Rap1-bound, and all nucleosomes. In panel E, −1 nucleosomes shared between divergently transcribed genes were removed, so as to clearly assess any asymmetry in Reb1 binding to nucleosome borders.
Figure 6
Figure 6. Srm1 Binds Nucleosomes Broadly and Pol II-bound Nucleosomes are Delocalized
The panels show the distribution of sequence tags for Srm1-bound (A–C) and Pol II-bound (D–F) nucleosomes for individual loci (A, D), a collection of genes (B, E in blue), and a composite profile (C, F blue trace), as described in Fig. 2. Rpo21 is the largest subunit of Pol II. The data shown in gold (E, F) represent the distribution of Pol II from standard sonication-based ChIP-chip.
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